PBL3 Antibody

What is PPIL3 and what biological functions does it serve?

PPIL3 (Peptidyl-prolyl cis-trans isomerase-like 3) belongs to the family of PPIases (peptidyl-prolyl isomerases). These enzymes accelerate protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . PPIL3 is also known by several other names including Cyclophilin J (CyPJ), Rotamase PPIL3, and Cyclophilin-like protein PPIL3 .

The primary functions of PPIL3 include:

• Facilitating protein folding through its PPIase activity

• Involvement in pre-mRNA splicing mechanisms

• Association with cyclophilin B through overlapping functions in protein folding and transport

Notably, PPIL3's functions in protein folding and transport can become impaired in neurodegenerative disorders, making it a protein of interest in neurological research .

How do I validate a PPIL3 antibody before using it in my experiments?

Proper validation of PPIL3 antibodies is critical for ensuring experimental reproducibility. Recent studies have shown that approximately 50% of commercial antibodies fail to meet basic standards for characterization , emphasizing the importance of thorough validation before use.

Based on current best practices, you should implement the following validation strategies:

• Genetic Validation Strategy: Use knockout or knockdown cell lines that do not express PPIL3 as negative controls. This is considered one of the gold standards for antibody validation .

• Orthogonal Strategy: Compare antibody-based measurements of PPIL3 with antibody-independent methods such as mass spectrometry or RNA sequencing to confirm correlation .

• Multiple Antibody Strategy: Test several independent antibodies targeting different epitopes of PPIL3 to verify consistent results .

• Recombinant Expression Strategy: Overexpress PPIL3 in a cell line and confirm increased signal with the antibody .

• Immunocapture MS Strategy: Use mass spectrometry to identify proteins captured by the PPIL3 antibody to confirm specificity .

For a PPIL3 antibody, validation should demonstrate that the antibody binds to PPIL3 and not to other proteins, recognizes PPIL3 in complex protein mixtures, and performs consistently in your specific assay conditions .

What applications is the PPIL3 antibody suitable for?

Based on available information about the PPIL3 antibody (ab169936), the following applications have been validated:

Confirmed Applications:

• Western Blotting (WB): The antibody has been tested and confirmed to work for human samples at a dilution of 1/500 .

Predicted Applications Based on Homology:
While not explicitly tested for all applications, based on protein homology and antibody characteristics, the PPIL3 antibody might be suitable for other common immunological techniques such as:

• Immunohistochemistry (IHC)

• Immunocytochemistry (ICC)

• Immunoprecipitation (IP)

• ELISA

Sample Compatibility:
The antibody has been specifically tested with:

• Human fetal brain lysate

• Human fetal liver lysate

Both showed the predicted band size of approximately 18 kDa in Western blot applications .

What positive and negative controls should I use when working with PPIL3 antibodies?

Appropriate controls are essential when working with any antibody, including those targeting PPIL3:

Recommended Positive Controls:

• Human Fetal Brain and Liver Lysates: These have been confirmed to express detectable levels of PPIL3 and can serve as positive controls in Western blotting applications .

• Recombinant PPIL3 Protein: Using purified recombinant PPIL3 protein can help verify antibody specificity and determine detection limits.

• Cells with Known PPIL3 Expression: Cell lines with documented PPIL3 expression levels can serve as reliable positive controls.

Recommended Negative Controls:

• PPIL3 Knockout/Knockdown Cells: Cells where PPIL3 has been genetically deleted or suppressed represent the gold standard negative control .

• Pre-absorption Control: Pre-incubating the antibody with purified PPIL3 protein should eliminate specific staining in subsequent assays.

• Isotype Control: Using an irrelevant antibody of the same isotype helps identify non-specific binding due to antibody class.

• Secondary Antibody Only: Omitting the primary antibody helps identify background from the detection system.

Recent comprehensive validation studies have emphasized that knockout/knockdown controls are particularly valuable, as they can reveal off-target binding that might otherwise go undetected with traditional controls .

What is the difference between monoclonal and polyclonal PPIL3 antibodies?

Understanding the differences between monoclonal and polyclonal antibodies is crucial when selecting the appropriate PPIL3 antibody for your research:

Polyclonal PPIL3 Antibodies:

• Generation: Produced by multiple B cell clones in an immunized animal (often rabbit)

• Epitope Recognition: Recognize multiple epitopes on the PPIL3 protein

• Advantages:

  • More robust to minor changes in the protein (denaturation, polymorphisms)

  • Often give stronger signals due to binding to multiple epitopes

  • Generally less expensive and faster to produce

• Disadvantages:

  • Batch-to-batch variability

  • Less specificity

  • Higher potential for cross-reactivity

• Example: The ab169936 PPIL3 antibody is a rabbit polyclonal antibody

Monoclonal PPIL3 Antibodies:

• Generation: Produced by a single B cell clone, usually through hybridoma technology

• Epitope Recognition: Recognize a single epitope on the PPIL3 protein

• Advantages:

  • Consistent batch-to-batch reproducibility

  • Higher specificity

  • Reduced background

• Disadvantages:

  • May be more sensitive to protein modifications affecting the specific epitope

  • Generally more expensive and time-consuming to produce

  • May have lower affinity compared to polyclonal antibodies

Recombinant Antibodies:
Recent studies have shown that recombinant antibodies (derived from synthetic genes) often outperform traditional monoclonal and polyclonal antibodies in terms of specificity and reproducibility . According to a comprehensive third-party testing of 614 commercial antibodies, recombinant antibodies generally demonstrated superior performance compared to traditional antibodies .

How can I troubleshoot non-specific binding when using PPIL3 antibodies?

Non-specific binding is a common challenge when working with antibodies. Here are methodological approaches to troubleshoot this issue:

Strategies to Reduce Non-specific Binding:

• Optimize Antibody Concentration:

  • Perform a dilution series to identify the optimal antibody concentration

  • For the PPIL3 antibody ab169936, the recommended dilution for Western blotting is 1/500, but this may need adjustment for your specific sample

• Improve Blocking Conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum)

  • Increase blocking time or concentration

  • Consider adding blocking agents to antibody diluent

• Increase Washing Stringency:

  • Add more washing steps

  • Increase detergent concentration (e.g., Tween-20, Triton X-100)

  • Extend washing times

• Pre-absorb the Antibody:

  • Incubate the antibody with tissue/cells known to cause cross-reactivity

  • For recombinant fragment antibodies like ab169936, consider pre-absorbing with tissue not expressing the N-terminal portion of PPIL3

• Modify Buffer Conditions:

  • Adjust salt concentration

  • Optimize pH

  • Add protein carriers or detergents

• Validate with Knockout Controls:

  • Test the antibody on PPIL3 knockout samples to identify non-specific bands or staining

  • This approach has been shown to be particularly effective in identifying antibody specificity issues

Common Sources of Non-specific Binding with PPIL3 Antibodies:

• Cross-reactivity with other cyclophilin family members

• Fc receptor binding, particularly in immune cells or tissues

• Endogenous peroxidase or phosphatase activity

• Protein-protein interactions due to PPIL3's role in protein folding

Remember that an antibody may exhibit dual specificity or cross-reactivity due to similarities in epitope structure or chemical composition . Therefore, thorough validation in your specific experimental system is essential.

What are the optimal conditions for using PPIL3 antibodies in various experimental techniques?

Optimizing experimental conditions is critical for obtaining reliable results with PPIL3 antibodies:

Western Blotting Conditions:

• Sample Preparation:

  • Use standardized lysis buffers with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Protein Loading:

  • 10-20 μg total protein per lane for cell lysates

  • Example: Human fetal brain and liver lysates have been successfully used

• Antibody Dilution:

  • 1/500 dilution for PPIL3 ab169936

• Detection System:

  • HRP-conjugated or fluorescent secondary antibodies

• Expected Results:

  • Band at approximately 18 kDa (predicted molecular weight of PPIL3)

Immunohistochemistry/Immunocytochemistry Considerations:

• Fixation:

  • 4% paraformaldehyde (PFA) or 10% neutral buffered formalin

  • Methanol fixation may better preserve protein epitopes

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

  • Protease-induced epitope retrieval as an alternative

• Blocking:

  • 5-10% normal serum from the same species as the secondary antibody

  • 1-3% BSA in PBS or TBS with 0.1-0.3% Triton X-100

• Antibody Incubation:

  • Primary: Overnight at 4°C

  • Secondary: 1-2 hours at room temperature

• Controls:

  • Include PPIL3 knockout or knockdown samples as negative controls

Immunoprecipitation Optimization:

• Lysis Buffer:

  • RIPA or NP-40 buffer with protease inhibitors

• Antibody Amount:

  • 2-5 μg per 500 μg of total protein

• Pre-clearing:

  • Pre-clear lysate with protein A/G beads to reduce background

• Washes:

  • 4-5 stringent washes in high-salt buffer

General Considerations:

• Temperature: Most antibody-antigen interactions are optimal at 4°C for extended incubations

• pH: Typically, neutral pH (7.2-7.4) works best

• Ionic Strength: Moderate salt concentration (150 mM NaCl) generally provides a good balance

• Additives: Low concentrations of detergents (0.05-0.1% Tween-20) can reduce non-specific interactions

Remember that antibody performance is context-dependent, and conditions may need to be optimized for each specific application and sample type .

How does PPIL3 antibody performance compare across different tissue/cell types?

Antibody performance can vary significantly across different tissues and cell types due to factors such as protein expression levels, post-translational modifications, and matrix effects:

PPIL3 Expression Patterns and Antibody Performance:

Confirmed Tissue Types for PPIL3 Antibody (ab169936):

• Human Fetal Brain: Successfully detected PPIL3 in Western blotting

• Human Fetal Liver: Successfully detected PPIL3 in Western blotting

Considerations for Tissue/Cell Type Variations:

Factors Affecting Tissue-Specific Performance:

• Epitope Accessibility: The accessibility of the PPIL3 epitope can vary between tissues due to differences in protein interactions or conformations

• Post-translational Modifications: Tissue-specific PTMs may alter antibody recognition of PPIL3

• Sample Preparation Effects: Different fixation and extraction methods can impact epitope preservation

• Background Interference: Endogenous peroxidases, phosphatases, or biotin can create tissue-specific background

• Matrix Effects: Complex tissue matrices can interfere with antibody binding

Recent studies have emphasized that antibody characterization is potentially cell or tissue type specific . Therefore, it is essential to validate the PPIL3 antibody in each new tissue or cell type, ideally using genetic controls (knockout/knockdown) when possible.

What are the implications of PPIL3's role in protein folding for neurodegenerative disorder research?

PPIL3's involvement in protein folding and transport processes makes it particularly relevant to neurodegenerative disorder research:

PPIL3 Function in Protein Folding and Neurodegenerative Disorders:

PPIL3 is associated with cyclophilin B through their overlapping functions in protein folding and transport, processes that can become impaired in neurodegenerative disorders . As a PPIase, PPIL3 catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, which is often a rate-limiting step in protein folding .

Potential Research Applications and Implications:

1. Protein Misfolding Studies:

• PPIL3 antibodies can be used to investigate changes in PPIL3 expression or localization in neurodegenerative disease models

• Changes in PPIL3 levels might correlate with protein misfolding severity

• Potential to identify upstream events in protein aggregation pathways

2. Therapeutic Target Investigation:

• Monitoring PPIL3 activity or expression as a potential biomarker

• Investigating whether modulation of PPIL3 activity could reduce protein misfolding

• Examining interactions between PPIL3 and known disease-associated proteins

3. Stress Response Pathways:

• PPIL3 may play a role in cellular stress responses related to protein folding

• Antibodies can help map changes in PPIL3 distribution during stress conditions

• Potential involvement in ER stress and unfolded protein response

Experimental Design Considerations:

Given the critical importance of protein folding in neurodegenerative disorders and PPIL3's function in this process, properly validated PPIL3 antibodies represent valuable tools for investigating disease mechanisms and potentially identifying novel therapeutic approaches.

How can I differentiate between true PPIL3 signal and artifacts in immunostaining?

Distinguishing true PPIL3 signal from artifacts is crucial for obtaining reliable immunostaining results:

Essential Controls for Validating PPIL3 Immunostaining:

• Genetic Controls (Gold Standard):

  • Use PPIL3 knockout or knockdown samples as negative controls

  • This approach has been identified as one of the most reliable validation methods

  • Allows clear differentiation between specific and non-specific signals

• Peptide Competition/Blocking Controls:

  • Pre-incubate PPIL3 antibody with purified PPIL3 protein or immunizing peptide

  • True PPIL3 signal should be eliminated or significantly reduced

  • Persistent signal indicates non-specific binding

• Multiple Antibody Validation:

  • Use independent antibodies targeting different PPIL3 epitopes

  • Concordant staining patterns suggest true signal

  • Discordant patterns warrant further investigation

• Technical Controls:

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control (irrelevant antibody of same isotype and concentration)

  • Endogenous enzyme blocking controls (for HRP or AP-based detection systems)

Pattern Recognition and Artifact Identification:

Advanced Validation Approaches:

• Orthogonal Validation:

  • Compare antibody-based PPIL3 detection with mRNA localization (e.g., in situ hybridization)

  • Correlation between protein and mRNA supports true signal

• Expected Biological Distribution:

  • Compare observed staining pattern with known PPIL3 expression patterns

  • Unexpected localization may indicate artifacts

Recent comprehensive studies have shown that thorough validation is essential, as approximately 50% of commercial antibodies fail to meet basic standards for characterization . Implementing multiple validation strategies is therefore critical for distinguishing true PPIL3 signal from artifacts.

What strategies can be employed for epitope mapping of PPIL3 antibodies?

Epitope mapping is essential for understanding exactly which part of the PPIL3 protein your antibody recognizes:

Epitope Mapping Strategies for PPIL3 Antibodies:

1. Peptide Array Mapping:

• Methodology:

  • Synthesize overlapping peptides spanning the entire PPIL3 sequence

  • Immobilize peptides on a membrane or chip

  • Probe with the PPIL3 antibody

  • Detect binding to identify the epitope region

• Advantages:

  • High resolution mapping

  • Can identify linear epitopes with precision

• Limitations:

  • Less effective for conformational epitopes

  • May miss contributions from distant residues

2. Deletion/Truncation Mapping:

• Methodology:

  • Create a series of PPIL3 truncation or deletion mutants

  • Express and purify the mutant proteins

  • Test antibody binding to each mutant

  • Loss of binding indicates epitope location

• Advantages:

  • Works for both linear and conformational epitopes

  • Provides information about minimum binding region

• Limitations:

  • Labor-intensive

  • Lower resolution than peptide arrays

3. Site-Directed Mutagenesis:

• Methodology:

  • Introduce point mutations in PPIL3 at suspected epitope residues

  • Express mutant proteins

  • Test antibody binding

  • Identify critical residues for antibody recognition

• Advantages:

  • Precise identification of critical binding residues

  • Can verify both linear and conformational epitopes

• Limitations:

  • Requires prior knowledge or prediction of potential epitope regions

  • Time-consuming

4. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Methodology:

  • Measure hydrogen-deuterium exchange rates in PPIL3 alone vs. antibody-bound

  • Regions protected from exchange when antibody is bound represent the epitope

• Advantages:

  • Works for conformational epitopes

  • No mutagenesis required

  • High resolution

• Limitations:

  • Requires specialized equipment

  • Complex data analysis

Application to PPIL3 Antibody (ab169936):

For the specific PPIL3 antibody mentioned in the search results (ab169936), we know it was generated using an immunogen corresponding to a recombinant fragment within human PPIL3 amino acids 1-150 . This indicates:

• The epitope is located within the N-terminal 150 amino acids of PPIL3

• Initial mapping could focus on this region

• The specific epitope is likely a linear sequence, as recombinant fragments were used

Understanding the precise epitope can help predict potential cross-reactivity with other cyclophilin family members, design blocking peptides for specificity controls, and interpret results when post-translational modifications occur near the epitope.

How can I measure the affinity of PPIL3 antibody-antigen interactions?

Measuring the affinity of PPIL3 antibody-antigen interactions is crucial for understanding binding strength, optimizing experimental conditions, and comparing different antibodies:

Methods for Measuring PPIL3 Antibody Affinity:

1. Surface Plasmon Resonance (SPR):

• Methodology:

  • Immobilize either PPIL3 protein or the antibody on a sensor chip

  • Flow the binding partner over the surface

  • Measure real-time association and dissociation

  • Calculate affinity constants (KD, ka, kd)

• Advantages:

  • Label-free detection

  • Real-time kinetics

  • Requires small amounts of material

• Equipment:

  • Commercial platforms like BIACore or IBIS as mentioned in the search results

• Expected Data:

  • Affinity (KD) typically in the nM to pM range for good antibodies

  • Association rate (ka): ~10^4-10^6 M^-1s^-1

  • Dissociation rate (kd): ~10^-4-10^-1 s^-1

2. Bio-Layer Interferometry (BLI):

• Methodology:

  • Similar to SPR but uses optical interference patterns

  • Immobilize PPIL3 or antibody on biosensors

  • Dip into solutions containing the binding partner

  • Measure wavelength shifts to determine binding

• Advantages:

  • No microfluidics required

  • Higher throughput than SPR

  • Less sensitive to buffer changes

• Limitations:

  • Generally less sensitive than SPR

3. Enzyme-Linked Immunosorbent Assay (ELISA):

• Methodology:

  • Coat plates with PPIL3

  • Add varying concentrations of antibody

  • Detect bound antibody

  • Plot binding curve and calculate apparent KD

• Advantages:

  • Accessible technology

  • No specialized equipment needed

  • High throughput

• Limitations:

  • Equilibrium measurements only (no kinetics)

  • Less precise than biophysical methods

Interpretation and Application of Affinity Data:

Practical Considerations for PPIL3 Antibody Affinity Measurement:

• Antigen Preparation:

  • Use properly folded, purified PPIL3 protein

  • Consider testing both full-length and the immunizing fragment (aa 1-150 for ab169936)

  • Verify protein quality by SDS-PAGE

• Antibody Preparation:

  • Purify antibody to remove contaminants

  • Determine accurate concentration

  • Use fresh preparations to avoid aggregation

Understanding the affinity of your PPIL3 antibody can help optimize experimental conditions, especially for applications like immunoprecipitation, ChIP, or flow cytometry where binding strength significantly impacts results .